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Am J Physiol Heart Circ Physiol 291: H2790-H2800, 2006. First published July 21, 2006; doi:10.1152/ajpheart.00535.2006
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Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Harjot K. Saini and Naranjan S. Dhalla

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 24 May 2006 ; accepted in final form 14 July 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although the Na+/H+ exchanger (NHE) is considered to be involved in regulation of intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger, the exact mechanisms of its participation in Ca2+ handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca2+ movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca2+]i in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca2+]i and augmented the KCl-induced increase in [Ca2+]i in a concentration-dependent manner. The MIA-induced increase in basal [Ca2+]i was unaffected by extracellular Ca2+, antagonists of the sarcolemmal (SL) L-type Ca2+ channel, and inhibitors of the SL Na+/Ca2+ exchanger, SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. However, the MIA-induced increase in basal [Ca2+]i was attenuated by inhibitors of SL Na+-K+-ATPase and sarcoplasmic reticulum (SR) Ca2+ transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca2+ concentration and attenuated by agents that inhibit SL L-type Ca2+ channels, the SL Na+/Ca2+ exchanger, SL Na+-K+-ATPase, and SR Ca2+ release channels and the SR Ca2+ pump. However, the effect of MIA on the KCl-induced increase in [Ca2+]i remained unaffected by treatment with inhibitors of SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca2+]i, whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca2+]i in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca2+]i and that MIA-induced increases in basal [Ca2+]i, as well as augmentation of the KCl-induced increase in [Ca2+]i, in cardiomyocytes are regulated differentially.

sodium-potassium adenosinetriphosphatase; calcium-handling proteins; sarcolemmal calcium transport; sarcoplasmic reticulum calcium transport; 5-(N-methyl-N-isobutyl) amiloride

IT IS WELL KNOWN that Ca2+ plays a critical role in cardiac excitation-contraction coupling, regulation of myocardial metabolism, and maintenance of cardiac cell integrity (6). Intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes is mainly regulated by sarcolemmal (SL) and sarcoplasmic reticulum (SR) proteins, and the mitochondrion and nucleus are also considered to participate in this process to some extent (6, 17, 42). SL L-type Ca2+ channels are the major pathways of Ca2+ entry, whereas SL Ca2+ pump ATPase is involved in Ca2+ efflux from cardiomyocytes. The SL Na+/Ca2+ exchanger has been suggested to participate in Ca2+ entry and Ca2+ removal, whereas the SL Na+ pump (Na+-K+-ATPase) is considered to regulate [Ca2+]i in cardiomyocytes indirectly through participation of the SL Na+/Ca2+ exchanger (6). In addition, SR Ca2+ ryanodine receptors are associated with Ca2+ release from the SR stores, whereas Ca2+ in SR stores is restored by SR Ca2+ pump ATPase (44). Involvement of the SL Na+/H+ exchanger (NHE) in development of intracellular Ca2+ overload has been suggested in different pathological conditions (3, 11). It is generally considered that activation of SL NHE by H+ in the myocardium leads to accumulation of Na+, which in turn increases [Ca2+]i through the SL Na+/Ca2+ exchanger (11). However, neither the participation of other Ca2+-regulating sites, such as SL Ca2+ channels and SR Ca2+ stores, nor the exact mechanisms of NHE-mediated alterations in [Ca2+]i in cardiomyocytes are completely understood. The present study was undertaken to test the hypothesis that changes in [Ca2+]i on alteration of NHE are mediated through participation of the SL Na+/Ca2+ exchanger and L-type Ca2+ channels, as well as SR Ca2+-regulating sites. To examine the participation of NHE in the mobilization of [Ca2+]i in quiescent, as well as KCl-depolarized, cardiomyocytes, Moffat and Karmazyn (26) blocked the exchanger with 5-(N-methyl-N-isobutyl)amiloride (MIA), a known inhibitor of NHE. Cardiomyocytes were treated with agents that are known to modify Ca2+ transport in SL, SR, and mitochondria to investigate the interaction of these agents with MIA to study Ca2+ mobilization. Because low Na+ has been shown to stimulate SL Na+/Ca2+ exchange activity in cardiomyocytes (37), some experiments were performed in the presence of low Na+ to investigate the direct contribution of the Na+/Ca2+ exchanger in the NHE-mediated modulation of [Ca2+]i. Because NHE is known to promote the efflux of H+ generated by myocardial metabolism (33, 36), pH of isolated cardiomyocytes was measured in the absence and presence of MIA.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of cardiomyocytes. Ventricular myocytes were isolated as described previously (40). Briefly, male Sprague-Dawley rats (250–300 g body wt) were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The hearts were quickly excised, mounted on a Langendorff apparatus, and perfused for 5 min with Ca2+-free buffer containing (in mM) 90 NaCl, 10 KCl, 1.2 KH2PO4, 5 MgSO4, 15 NaHCO3, 30 taurine, and 20 glucose and gassed with 95% O2-5% CO2 at 37°C, pH 7.4. The hearts were then switched to the same perfusion medium containing 0.04% collagenase, 0.1% BSA, and 50 µM CaCl2. At the end of a 25-min recirculation period, perfusion was stopped. The ventricles were cut into small pieces and subjected to 15 min of digestion in a fresh collagenase solution containing 1% BSA gassed with 95% O2-5% CO2 in a shaking water bath at 37°C. The ventricular fragments were triturated gently (twice per minute) with a plastic pipette. The cells from three to four harvests were combined and filtered through 200-µm nylon mesh. The cardiomyocytes were resuspended for 5 min in buffers containing gradually increasing extracellular Ca2+ (250, 500, and 750 µM) to a final concentration of 1 mM. Cell viability in all experimental groups was determined by the trypan blue (Sigma-Aldrich, Oakville, ON, Canada) exclusion method. A Neubauer chamber was used to count the number of unstained and stained cells and the total number of cells. The final cell suspension consisted of 80–85% viable, quiescent cardiomyocytes; 3–5% of the cardiomyocytes beat spontaneously. All protocols were approved by the University of Manitoba Animal Care Committee and were conducted in accordance with the standards of the Canadian Council on Animal Care.

Measurement of [Ca2+]i. Freshly isolated cardiomyocytes were incubated with 5 µM fura 2-AM for 40 min in a buffer (pH 7.4) containing (in mM) 90 NaCl, 10 KCl, 1.2 KH2PO4, 5 MgSO4, 15 NaHCO3, 30 taurine, 20 glucose, and 1 CaCl2 and 1% BSA. The cells were washed twice with the same solution for removal of any extracellular dye. The final cell number in the cuvette was adjusted to 3 x 105 cells/ml for all the experimental groups. Alterations in fluorescence intensity were monitored by a dual-wavelength spectrofluorometer (model DMX-1100, SLM Instruments, Urbana, IL) adjusted to 340/380-nm excitation, 510-nm emission, 0.95-s integration time, and 1.0-s resolution time. [Ca2+]i was calculated as described previously (40). In some experiments, the cells were pretreated with MIA for 10 min at room temperature before measurement of fluorescence in the presence of KCl or ATP. In all other experiments, fura 2-loaded cells inside the cuvette were treated with different concentrations of MIA for 10 min. No photobleaching of the fluorometric recording was observed during this time period. Modulation of [Ca2+]i was determined by incubation of the fura 2-loaded cells in buffer containing the desired concentration of pharmacological agents [except ryanodine and cyclopiazonic acid (CPA)] for 10 min before measurement of fluorescence in the presence and absence of MIA; the cells were treated with ryanodine and CPA for 20 min before determination of [Ca2+]i. The concentrations of different pharmacological agents were selected on the basis of our previous experience (37, 41). The effect of low Na+ on MIA-mediated alterations in [Ca2+]i was examined by treatment of the cardiomyocytes with Krebs-Henseleit buffer (pH 7.4) containing 70 or 35 mM extracellular Na+ for 10 min at room temperature; osmolarity of the solution was maintained by addition of choline chloride, as described elsewhere (41). Treatment with the various pharmacological agents under incubation conditions did not change cell viability. The increase in [Ca2+]i at peak [Ca2+]i was calculated as the net increase above the basal value in each experiment. The difference between the responses in the absence and presence of MIA was taken as the MIA-induced increase in [Ca2+]i.

Measurement of intracellular pH. The protocol for measurement of intracellular H+ concentration was identical to that described by others (21). Briefly, isolated cardiomyocytes were loaded with 4 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. A calibration curve of BCECF-loaded cardiomyocytes was plotted by measuring the ratio of fluorescence at 440-nm excitation to fluorescence at 500-nm excitation and recording emission at 525 nm with different standard solutions (pH 5.5–7.4) containing high K+ and 10 µM nigericin, a known H+-K+ ionophore (21). No quenching effect of BCECF-AM fluorescence was observed at pH 5.5–7.4. Alterations in intracellular pH induced by different concentrations of MIA were measured in experiments performed in HCO3–-free buffers, as described previously (39), to eliminate the activities of other alkalinizing mechanisms, such the Na+-HCO3– cotransporter and Cl–/HCO3– exchanger. In some experiments, pH was measured in the presence of HCO3–-containing buffer used for determination of [Ca2+]i to mimic the physiological conditions.

Statistical analysis. Values are means ± SE. Statistical analysis was performed with Microcal Origin version 6 (Microcal Software, Northampton, MA). Differences between two groups were evaluated by Student’s t-test. Data from more than two groups were evaluated by one-way ANOVA followed by Newman-Keuls test. P < 0.05 was considered statistically significant unless otherwise indicated.

Drugs and chemicals. MIA, amiloride, nigericin, 5-(N,N)-dimethylamiloride (DMA), verapamil, diltiazem, vanadate, ouabain, sodium azide, ruthenium red, ryanodine, caffeine, and CPA were purchased from Sigma-Aldrich; KB-R7943 from Tocris Biosciences (Ellisville, MO); fura 2-AM and BCECF-AM from Molecular Probes (Eugene, OR); and collagenase (type II, 265 U/mg) from Worthington Biochemical (Freehold, NJ). All other reagents were of analytic grade and were purchased from Sigma-Aldrich or Fisher Scientific (Fair Lawn, NJ).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of MIA on [Ca2+]i in isolated cardiomyocytes. In the first set of experiments, isolated cardiomyocytes were pretreated with 1–10 µM MIA, and basal [Ca2+]i and changes in [Ca2+]i on addition of 30 mM KCl, a known depolarizing agent, were monitored (40). Pretreatment of cells with 5 µM MIA increased basal [Ca2+]i and augmented the KCl-induced increase in [Ca2+]i (Fig. 1A). As shown in Fig. 1, B and C, the increase in basal [Ca2+]i and augmentation of the KCl-induced increase in [Ca2+]i were dependent on MIA concentration (1–10 µM). The specific nature of augmentation of the KCl-induced increase in [Ca2+]i was determined from the effect of MIA on [Ca2+]i in the presence of ATP, a purinergic receptor agonist (40). The representative trace in Fig. 1D shows the effect of 5 µM MIA on basal and ATP responses. Pretreatment of the cells with 1–10 µM MIA increased basal [Ca2+]i and augmented the ATP-induced increase in [Ca2+]i (Fig. 1, E and F).


Figure 1
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  		Fig. 1. Effect of 1–10 µM 5-(N-methyl-N-isobutyl)amiloride (MIA) on basal intracellular Ca2+ concentration ([Ca2+]i), as well as KCl (30 mM)- and ATP (50 µM)-mediated increase in [Ca2+]i, in isolated cardiomyocytes. A: effect of 5 µM MIA on basal and KCl-mediated increase in [Ca2+]i. B: effect of 0.25–10 µM MIA on basal [Ca2+]i before addition of KCl. C: effect of 0.25–10 µM MIA on KCl-mediated increase in [Ca2+]i. D: effect of 5 µM MIA on basal and ATP-mediated increase in [Ca2+]i. E: effect of 0.25–10 µM MIA on basal [Ca2+]i before addition of ATP. F: effect of 0.25–10 µM MIA on ATP-mediated increase in [Ca2+]i. Values are means ± SE of 4 preparations in each group. *P < 0.05 vs. control (0 MIA).

 
In another set of experiments, we addressed the possibility that an artifact might be due to pretreatment of cardiomyocytes with MIA. The effects of MIA on [Ca2+]i were studied in the cuvette, and real-time traces were recorded. MIA increased basal [Ca2+]i in a concentration-dependent manner, similar to that observed with pretreatment of cardiomyocytes (Fig. 2A). Augmentation of the KCl-mediated increase in [Ca2+]i was observed at different MIA concentrations (Fig. 2, B, D, and F). The maximal increase in basal [Ca2+]i and MIA-induced augmentation of the KCl-mediated response were observed at 10 µM MIA (Fig. 2, C–F); further increase in MIA concentration resulted in no additional potentiation in these parameters. Because 5 µM MIA produced a substantial increase in basal [Ca2+]i, as well as augmentation of the KCl-induced response, all further experiments were performed using 5 µM MIA in the presence of different pharmacological interventions.


Figure 2
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  		Fig. 2. Effect of addition of 1–10 µM MIA to the cuvette on basal [Ca2+]i and KCl-mediated increase in [Ca2+]i in isolated cardiomyocytes. A: effect of 1–10 µM MIA on basal [Ca2+]i. B: effect of 5 µM MIA on basal and KCl-mediated increase in [Ca2+]i. C: effect of 1–10 µM MIA on basal [Ca2+]i. D: effect of 1–10 µM MIA on KCl-mediated increase in [Ca2+]i. E: dose response for MIA-induced increase in basal [Ca2+]i. F: dose response for MIA-induced augmentation of KCl response. MIA-induced increase in [Ca2+]i is the difference between basal or KCl-induced increase in [Ca2+]i in the absence and presence of MIA. Values are means ± SE of 4 experiments in each group. *P < 0.05 vs. control (0 MIA). #P < 0.05 vs. blank, which showed no increase.

 
To establish whether the effects of MIA on [Ca2+]i are simulated by amiloride or its other derivatives, changes in [Ca2+]i in cardiomyocytes were measured in the presence of amiloride, a nonspecific NHE inhibitor (37, 41), as well as DMA, a specific NHE inhibitor (10). Amiloride (5–20 µM) did not change basal [Ca2+]i but, in a concentration-dependent manner, significantly attenuated the KCl-induced increase in [Ca2+]i (Table 1). These findings are consistent with our previous observations under similar experimental conditions (41). Amiloride-induced alterations in [Ca2+]i do not represent the effects of NHE inhibition, inasmuch as amiloride is also known to inhibit SL Ca2+-regulating sites, such as the Na+/Ca2+ exchanger, Na+-K+-ATPase, Na+ channels, and T-type Ca2+ channels (34). On the other hand, DMA (5–20 µM) significantly increased basal [Ca2+]i and augmented the KCl-mediated increase in [Ca2+]i (Table 1). Because DMA had some effects on the autofluorescence of unloaded cardiomyocytes, no further experiments were performed in the presence of DMA. On the other hand, 5 µM MIA does not show any autofluorescence and, thus, does not interfere with fura 2 measurement of [Ca2+]i.


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  		Table 1. Effect of amiloride and DMA on basal [Ca2+]i and KCl-induced increase in [Ca2+]i in isolated cardiomyocytes

 
Effect of extracellular Ca2+ on the MIA-induced increase in [Ca2+]i. To examine the direct involvement of extracellular Ca2+ in the MIA-mediated increase in basal [Ca2+]i and augmentation of the KCl-mediated response, isolated cardiomyocytes were treated with low (0.5 mM) and high (2.5 mM) concentrations of extracellular Ca2+. Basal [Ca2+]i and the MIA-induced increase in basal [Ca2+]i remained unaltered at 0.5–2.5 mM extracellular Ca2+. On the other hand, the KCl-induced increase in [Ca2+]i and MIA-mediated augmentation of the KCl-mediated response in cardiomyocytes (1.25 mM extracellular Ca2+) were significantly depressed by decreasing extracellular Ca2+ to 0.5 mM (Table 2). On the other hand, increasing extracellular Ca2+ to 2.5 mM significantly potentiated the KCl-mediated increase in [Ca2+]i and augmented the MIA-induced increase in the KCl-mediated response (Table 2).


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  		Table 2. Effect of extracellular Ca2+ on basal [Ca2+]i and KCl-induced increase in [Ca2+]i in absence or presence of MIA in isolated cardiomyocytes

 
Modulation of the MIA-induced increase in [Ca2+]i by L-type Ca2+ channels. To investigate the involvement of extracellular Ca2+ in the MIA-induced increase in basal [Ca2+]i and augmentation of the KCl-mediated response, we treated isolated cardiomyocytes with two well-known L-type Ca2+ channel antagonists, verapamil and diltiazem (41). Preincubation of cells with verapamil (1 and 5 µM) or diltiazem (1 and 5 µM) did not significantly change basal [Ca2+]i or the MIA-mediated increase in basal [Ca2+]i (Table 3). On the other hand, the KCl-induced increase in [Ca2+]i and the MIA-mediated augmentation of the KCl-induced response were significantly attenuated by verapamil and diltiazem (Table 3).


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  		Table 3. Effect of verapamil and diltiazem on basal [Ca2+]i and KCl-induced increase in [Ca2+]i in absence or presence of MIA in isolated cardiomyocytes

 
Role of SL Ca2+ pump ATPase and Na+-K+-ATPase in the MIA-induced increase in [Ca2+]i. Involvement of SL Ca2+ pump ATPase and SL Na+-K+-ATPase in the MIA-induced increase in [Ca2+]i was studied by treatment of the cells with specific inhibitors of these enzymes. Preincubation with vanadate (0.5, 1, and 2 µM), a known inhibitor of SL Ca2+ pump ATPase (41), did not change basal [Ca2+]i or the MIA-mediated increase in basal [Ca2+]i. Similarly, vanadate did not alter the KCl-mediated increase in [Ca2+]i and the MIA-mediated augmentation of the KCl-induced response (Table 4). On the other hand, ouabain (0.05, 0.1, and 0.3 mM), a known inhibitor of Na+-K+-ATPase (20), significantly attenuated the MIA-mediated increase in basal [Ca2+]i and the KCl-induced response (Table 4). Ouabain at 0.05–0.3 mM significantly increased basal [Ca2+]i and augmented the KCl-mediated increase in [Ca2+]i. On the other hand, 0.01 mM ouabain did not change basal [Ca2+]i or the KCl-mediated increase in [Ca2+]i in the absence or presence of MIA (Table 4).


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  		Table 4. Effect of ouabain and vanadate on basal [Ca2+]i and KCl-induced increase in [Ca2+]i in absence or presence of MIA in isolated cardiomyocytes

 
Involvement of the Na+/Ca2+ exchanger in the MIA-induced increase in [Ca2+]i. To test the role of the Na+/Ca2+ exchanger in the MIA-induced increase in basal [Ca2+]i and the KCl-mediated response, the cells were treated with KB-R7943, a known inhibitor of the Na+/Ca2+ exchanger (41). KB-R7943 (10 and 25 µM) did not change basal [Ca2+]i or the MIA-induced increase in basal [Ca2+]i (Table 5). On the other hand, the KCl-induced increase in [Ca2+]i and MIA-mediated augmentation of the KCl-induced response were significantly attenuated by KB-R7943 (Table 5). Because low Na+ has been shown to increase [Ca2+]i by activating the SL Na+/Ca2+ exchanger, the cells were exposed to Krebs-Henseleit solution containing 70 or 35 mM Na+ before measurement of fluorescence. Low-Na+ solution did not depress the MIA-mediated increase in basal [Ca2+]i but markedly depressed the MIA-induced augmentation of the KCl-mediated response (Table 5). Low Na+ itself had no effect on basal [Ca2+]i but significantly potentiated the KCl-induced increase in [Ca2+]i.


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  		Table 5. Effect of KB-R7943 and low Na+ on basal [Ca2+]i and KCl-induced increase in [Ca2+]i in absence or presence of MIA in isolated cardiomyocytes

 
Involvement of mitochondrial Ca2+ regulatory mechanisms in the MIA-induced increase in [Ca2+]i. The role of mitochondria in the MIA-induced increase in [Ca2+]i was examined in cardiomyocytes treated with 0.5–10 mM sodium azide and 0.5–10 µM ruthenium red, inhibitors of mitochondrial Ca2+ uptake (8, 15). Neither of these agents affected basal [Ca2+]i or the KCl-induced increase in [Ca2+]i. Similarly, the MIA-mediated increase in basal [Ca2+]i (42 ± 3.4 nM for control, 40 ± 5.1 and 41 ± 3.3 nM for 0.5 and 1 mM sodium azide, respectively, and 42 ± 3.7 and 41 ± 4.0 nM for 0.5 and 1 µM ruthenium red, respectively) and augmentation of the KCl-mediated response (42 ± 3.9 nM for control, 46 ± 4.7 and 45 ± 4.3 nM for 0.5 and 1 mM sodium azide, respectively, and 41 ± 3.8 and 42 ± 4.7 nM for 0.5 and 1 µM ruthenium red, respectively) remained unaltered in the presence of both of these agents.

Role of SR Ca2+ stores in the MIA-induced increase in [Ca2+]i. To assess the involvement of SR Ca2+ stores in the MIA-mediated alterations in basal Ca2+ and the KCl-induced response, cardiomyocytes were treated with agents that are known to modulate SR Ca2+ stores. Preincubation of cells with ryanodine (2 and 5 µM), a blocker of the SR Ca2+ release channel (41), attenuated the MIA-mediated increase in basal [Ca2+]i and the KCl-induced increase in [Ca2+]i. Similarly, pretreatment of cells with CPA (20 and 50 µM), a known SR Ca2+ pump ATPase inhibitor (41), significantly decreased the MIA-induced increase in basal [Ca2+]i (Table 6). Representative traces in Fig. 3A show the effect of caffeine pretreatment on the MIA-induced increase in basal [Ca2+]i and augmentation of the KCl-mediated response. Caffeine (10 and 20 mM), which maintains the open state of Ca2+ release channels (45), attenuated the MIA-induced increase in basal [Ca2+]i (Fig. 3, B and D). In addition, MIA-mediated augmentation of the KCl-induced response was significantly depressed by treatment with these agents (Table 6, Fig. 3, C and D). Preincubation with ryanodine, CPA, and caffeine had no effect on basal [Ca2+]i, whereas the KCl-mediated increase in [Ca2+]i was significantly depressed (Table 6, Fig. 3). In contrast to a typical Ca2+ transient obtained on electrical stimulation of a single cardiomyocyte, the increase in [Ca2+]i after KCl addition, as measured in the preparation used in this study, did not decrease over time. The presence of KCl in the cuvette at all times during fluorescence measurement may be the reason for such a response, which is consistent with results from our previous studies (40, 41, 46).


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  		Table 6. Effect of ryanodine and CPA on basal [Ca2+]i and KCl-induced increase in [Ca2+]i in absence or presence of MIA in isolated cardiomyocytes

 

Figure 3
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  		Fig. 3. Effect of 5–20 mM caffeine on MIA (5 µM)-mediated increase in basal and KCl-induced augmentation of [Ca2+]i. A: effect of 20 mM caffeine on MIA-mediated increase in basal and KCl-induced augmentation of [Ca2+]i. B: effect of 5–20 mM caffeine on basal [Ca2+]i in the presence of MIA. C: effect of 5–20 mM caffeine on KCl-mediated increase in [Ca2+]i in the presence of MIA. D: dose response for caffeine showing effect on MIA-induced increase in basal [Ca2+]i. E: dose response for caffeine showing effect on MIA-induced augmentation of KCl-mediated increase in [Ca2+]i. MIA-induced increase in [Ca2+]i is the difference between basal or KCl-induced increase in [Ca2+]i in the absence and presence of MIA. Values are means ± SE of 4 preparations in each group. C, control cardiomyocytes (0 MIA or 0 caffeine). *P < 0.05 vs. control (MIA without caffeine).

 
Effect of lowering intracellular pH on [Ca2+]i. To understand the mechanisms of the MIA-induced increase in basal [Ca2+]i and augmentation of the KCl-mediated increase, we investigated the effects of extracellular pH and MIA on intracellular pH. Reduction of extracellular pH from 7.4 to 5.5 decreased intracellular pH (Fig. 4A). Reduction of pH to 5.5 significantly increased basal [Ca2+]i (Fig. 4B). On the other hand, reduction of extracellular pH from 7.4 to 6.5 did not significantly affect the KCl-induced increase in [Ca2+]i, which was significantly decreased at pH 6.0 and 5.5 (Fig. 4C). MIA (1–10 µM) significantly reduced pH in cardiomyocytes in HCO3–-free buffer (Fig. 4D) and increased basal [Ca2+]i under the experimental conditions used for pH measurements (Fig. 4E). A highly significant linear relation was observed for changes in basal [Ca2+]i and decrease in pH, in contrast to the KCl-induced increase in [Ca2+]i and decrease in pH in an acidic environment and in the presence of MIA (Fig. 5F). In another set of experiments using three separate preparations and HCO3–-containing buffer, intracellular pH was 7.12 ± 0.03 (control), 6.90 ± 0.10 (1 µM MIA), 6.32 ± 0.04 (5 µM MIA), and 5.95 ± 0.05 (10 µM MIA). These values in the presence of HCO3–-containing buffer were not qualitatively different from those in the absence of HCO3–-containing buffer.


Figure 4
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  		Fig. 4. Effect of extracellular pH and MIA on intracellular pH and increase in basal [Ca2+]i in isolated cardiomyocytes. A: effect of extracellular pH (5.5–7.4) on intracellular pH. B: effect of extracellular pH (5.5–7.4) on basal [Ca2+]i. C: effect of extracellular pH (5.5–7.4) on KCl-induced increase in [Ca2+]i. D: effect of 1–10 µM MIA on intracellular pH. E: effect of 1–10 µM MIA on basal [Ca2+]i under the same conditions used for determination of pH. F: correlation of intracellular pH and basal [Ca2+]i in isolated cardiomyocytes at different extracellular pH ({blacksquare}) and different MIA concentrations (bullet). Values are means ± SE of 4 experiments in each group. *P < 0.05 vs. 0 MIA. #P < 0.05 vs. extracellular pH 7.4.

 

Figure 5
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  		Fig. 5. Schematic representation of MIA-induced increase in basal [Ca2+]i, as well as augmentation of KCl-mediated increase in [Ca2+]i, in cardiomyocytes. Sarcolemma (SL) Ca2+ sites include mainly L-type Ca2+ channels and Na+/Ca2+ exchanger; sarcoplasmic reticulum (SR) Ca2+ stores include Ca2+ release channels and Ca2+ pump ATPase mechanisms. [H+]i, intracellular H+ concentration.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The results of the present study show that inhibition of NHE by MIA significantly increases basal [Ca2+]i. This increase in basal [Ca2+]i seems to be specific to the derivatives of amiloride, inasmuch as amiloride itself did not increase basal [Ca2+]i under similar experimental conditions, whereas DMA, a selective NHE inhibitor (10), significantly increases basal [Ca2+]i. The MIA-induced increase in basal [Ca2+]i appears to be independent of extracellular Ca2+ concentration, because inhibition of SL L-type Ca2+ channels or the Na+/Ca2+ exchanger did not affect the MIA-mediated increase in [Ca2+]i. In addition, a change in extracellular Ca2+ concentration did not alter the MIA-induced increase in basal [Ca2+]i. The MIA-mediated increase in basal [Ca2+]i was not changed by SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake inhibitors. On the other hand, involvement of SR Ca2+ stores in modifying the MIA-mediated increase in basal [Ca2+]i is apparent from the observation that depletion of SR Ca2+ stores by ryanodine, caffeine, and CPA attenuated the MIA-induced increase in basal [Ca2+]i in quiescent cardiomyocytes. Because reduction of intracellular pH increased basal Ca2+, as observed in the present study as well as previously (29, 35), it is likely that the MIA-mediated reduction in intracellular pH is responsible for such an increase in basal [Ca2+]i. Orchard et al. (30) showed that acidosis facilitates the spontaneous SR Ca2+ release in rat myocardium, which can be abolished by ryanodine and caffeine. Furthermore, acidosis has been shown to reduce SR Ca2+ uptake (9) and affect the L-type Ca2+ channels and Na+/Ca2+ exchange activity (12, 19). In view of the linear relation between the decrease in intracellular pH and basal [Ca2+]i observed in this study, it appears that the increase in basal [Ca2+]i by NHE inhibition may be a consequence of the decrease in pH with a subsequent release of Ca2+ from SR, as well as changes in SL Ca2+ transport. MIA was found to exert no direct effect on SR Ca2+ uptake or SR Ca2+ release under in vitro conditions (unpublished observations). Although the increase in basal [Ca2+]i induced by NHE inhibition may not represent a marked intracellular Ca2+ overload, it may affect the steady-state [Ca2+]i over time.

It is well documented that the KCl-mediated increase in [Ca2+]i is dependent on extracellular Ca2+ concentration and is mediated by SL and SR Ca2+-regulating sites (37, 46). The results of the present study show that the KCl-mediated increase in [Ca2+]i was augmented by MIA. Such an augmentation seems to be a consequence of changes in SL and SR Ca2+-regulating sites. This view is based on the observations that SL L-type Ca2+ channel blockers, Na+/Ca2+ exchange inhibitor, and SR Ca2+ store-modulating agents attenuate the MIA-mediated augmentation of the KCl-induced response. On the other hand, mitochondrial Ca2+ uptake and SL Ca2+ pump ATPase inhibitors did not prevent the KCl-induced increase in [Ca2+]i or the MIA-mediated potentiation of the KCl-induced response. Cui et al. (5) showed that DMA, another NHE inhibitor, increased the Ca2+ transient and cell shortening in isolated cardiomyocytes, which are mediated by stimulation of the Na+/Ca2+ exchanger without involvement of Ca2+ entry through L-type Ca2+ channels. The differences in the regulation of [Ca2+]i in electrically stimulated Ca2+ transients and KCl-depolarized cardiomyocytes may be the reason for this discrepancy. Extracellular K+ has been reported to inhibit NHE-1, the most abundant NHE isoform in cardiomyocytes (31); thus participation of such an inhibition by KCl under the experimental conditions in the present study cannot be ruled out. Also, the KCl-induced increase in [Ca2+]i, in contrast to basal [Ca2+]i, was depressed by decreasing extracellular pH from 6.0 to 5.5. This is consistent with the decrease in the Ca2+ transient due to acidosis (29). A schematic diagram recapitulating the mechanisms of MIA-mediated alterations in [Ca2+]i in cardiomyocytes (Fig. 5) shows the differences in the regulation of basal [Ca2+]i and KCl-induced changes in [Ca2+]i in cardiomyocytes. Nonetheless, the present study suggests that MIA-mediated augmentation of the KCl-induced response may be mediated by changes in Ca2+ handling at SL and SR Ca2+-regulating sites.

Ouabain, an Na+-K+-ATPase inhibitor (20), markedly increased basal, as well as KCl-mediated, augmentation of [Ca2+]i. These results are consistent with our previous observations (37). Because ouabain is known to increase [Ca2+]i by promoting entry of Ca2+ through the Na+/Ca2+ exchanger (20), it was expected that the MIA-induced increase in [Ca2+]i would be depressed by ouabain. This was found to be the case in the present study, indicating the interaction of ouabain with MIA. Nakanishi et al. (28) also showed attenuation of the ouabain-induced increase in [Ca2+]i after NHE inhibition. Bolck et al. (1) demonstrated that Na+/Ca2+ exchanger overexpression reduces cell shortening at higher stimulation frequencies and after inhibition of Na+-K+-ATPase by ouabain. Furthermore, low Na+, which is known to enhance Na+/Ca2+ exchanger activity (37), did not affect the MIA-induced increase in basal [Ca2+]i but did depress MIA-mediated augmentation of the KCl-induced response. Thus it appears that the interaction of MIA and ouabain in the KCl-induced increase in [Ca2+]i may be a consequence of the direct action of ouabain on SL Na+-K+-ATPase and the indirect action of MIA on the SL Na+/Ca2+ exchanger. The contribution of alterations in intracellular pH cannot be ruled out, inasmuch as ouabain has been shown to significantly decrease pH in the presence and absence of an NHE inhibitor (20).

The results of the present study suggest that MIA-mediated alterations in [Ca2+]i mobilization are regulated differently in quiescent and depolarized cardiomyocytes. Although NHE is an important SL site for promoting the efflux of H+ and, thus, leads to the decrease of metabolic acidosis due to its inhibition by agents such as MIA (33, 43), its contribution under normal physiological conditions is controversial (4, 7, 18). Kusuoka et al. (16) demonstrated that NHE works actively in the physiological range of pH in isovolumically contracting hearts and that blockade of this exchanger decreases pH under steady-state conditions. Prolonged inhibition of NHE by MIA with a significant decrease in pH, as observed previously (26) and in the present study, indicates that this exchanger is active under the conditions employed here. Because we used a pharmacological approach to obtain some information about the role of NHE in regulating [Ca2+]i in cardiomyocytes, further experiments employing molecular approaches are needed to investigate the involvement of a particular site in NHE-mediated alterations in [Ca2+]i. Studies employing site-specific mutagenesis and transgenic animals with NHE knockout may further unravel the mechanisms for such an effect of NHE inhibition on Ca2+ handling by cardiomyocytes. Although it can be argued that the Ca2+-handling experiments in the present study were conducted in the presence of buffer containing HCO3– and, thus, the changes may not be the true reflection of NHE inhibition in cardiomyocytes, changes in basal [Ca2+]i and MIA-mediated augmentation of the KCl-induced response, as well as MIA-induced alterations in pH, were qualitatively similar in the absence and presence of HCO3–-containing buffer. Ruiz-Meana et al. (38) also showed that metabolic inhibition-mediated increase in intracellular Ca2+ and Na+ in the presence of HOE-642 (cariporide), a selective NHE inhibitor, was independent of HCO3– in the perfusion medium.

MIA was selected for the present study because it is available and does not interfere with fura 2 excitation and emission wavelengths. MIA (1–10 µM) is considered to inhibit NHE specifically, inasmuch as its IC50 for NHE was 14 µM, whereas its IC50 values for the Na+/Ca2+ exchanger, Ca2+ pump, and Na+ pump were 84, 70, and >300 µM, respectively (27). Because DMA, another specific inhibitor of NHE (10), interferes with fura 2, the values for basal [Ca2+]i and the KCl-induced [Ca2+]i response due to this agent were corrected appropriately. Similar interference in fura 2 excitation wavelength was observed in the previous study (39) in the presence of ethylisopropylamiloride, which has a 10-fold higher potency than DMA to block NHE (13). Although cardiotoxicity of amiloride derivatives has limited their use as an experimental tool (27), no such effect of MIA on cardiomyocyte viability was observed in the present study. Nonetheless, MIA-induced intracellular Ca2+ overload in cardiomyocytes, as observed in this study, can be considered to explain the cardiotoxic effects of high concentrations of amiloride derivatives (27). Other investigators used benzoylguaninidine derivatives (cariporide) and bicyclic guanidines (zoniporide, SMP-300, SM-20220, and SM-20550) as NHE inhibitors (22) to study their effects on cardiac function but obtained varying results. Such differences in results seem to depend on the animal model, optimal dose, timing of drug delivery, and disparity in selection of the end point (10, 11). However, the results of the clinical trials with some NHE inhibitors in patients with ischemic heart disease were not promising (14). Nonetheless, extrapolation of the MIA-induced increase in basal [Ca2+]i and augmentation of the KCl-mediated increase in [Ca2+]i in cardiomyocytes to the cerebrovascular system suggests vasoconstriction and cerebral spasm in the brain on inhibition of the SL NHE. Elevated Ca2+ in the brain has been shown to produce neuronal death (24). Intracellular Ca2+ overload can also have deleterious effects on cardiac and vascular cells over a prolonged period of treatment with NHE inhibitors (2, 25). In addition, the MIA-induced decrease in intracellular pH also indicates the potential for NHE inhibitors to produce intracellular acidosis, which may be detrimental to cardiac function.

Because we did not compare the effects of MIA with those of some of the newer NHE inhibitors such as cariporide, it is difficult to comment on the clinical significance of the results presented in this study. However, cariporide has been reported not to depress the anoxia-induced intracellular Ca2+ overload (39). Similarly, the metabolic inhibition-mediated increase in intracellular Na+ and Ca2+ was not prevented by cariporide (38). On the other hand, chemical hypoxia-mediated Ca2+ accumulation was attenuated by cariporide (32). Although cariporide had a beneficial effect on heart function in a clinical trial (23), it increased the incidence of stroke. Thrombotic stroke and platelet aggregation in cariporide-treated patients (23) were apparently not identified as effects of NHE inhibitors. Thus a great deal of caution should be exercised in interpretation of the observations with MIA in this study in terms of explaining the potential toxicity of the newer NHE inhibitors.


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The work was supported by a grant from the Canadian Institutes of Health Research. H. K. Saini is a predoctoral fellow of the Heart and Stroke Foundation of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (e-mail: nsdhalla{at}sbrc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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H. K. Saini and N. S. Dhalla
Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H169 - H181.
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